Research Article |
Corresponding author: Watchariya Purivirojkul ( fsciwyp@ku.ac.th ) Academic editor: Fredric Govedich
© 2019 Poramad Trivalairat, Krittiya Chiangkul, Watchariya Purivirojkul.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Trivalairat P, Chiangkul K, Purivirojkul W (2019) Placobdelloides sirikanchanae sp. nov., a new species of glossiphoniid leech and a parasite of turtles from lower southern Thailand (Hirudinea, Rhynchobdellida). ZooKeys 882: 1-24. https://doi.org/10.3897/zookeys.882.35229
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Abstract A new species of glossiphoniid leech, Placobdelloides sirikanchanae sp. nov., is reported in the Asian leaf turtle (Cyclemys dentata) and the dark-bellied leaf turtle (C. enigmatica) from Songkhla Province, southern Thailand. The examination of morphological characters revealed that this new species is similar to P. siamensis (Oka, 1917), a common turtle leech species found in Thailand. Placobdelloides sirikanchanae sp. nov. demonstrates distinct morphological characters, with an elongated, narrow body, 13–17 well-developed knob papillae on each annulus, dark brown to greenish dorsal color with a crimson median line, the absence of a scarlet dot, different male and female gonopore distributions, a rough posterior sucker with a random pit distribution, and 104–115 eggs per clutch. The phylogenetic relationships of COI-ND1 genes were clarified and shown to be distinct from those of P. siamensis. Additionally, habitat preferences tended toward low oxygen conditions such as puddles or water patches on rubber plantations.
Clitellata, Cyclemys, Glossiphoniidae, Hirudinea, leaf turtle, Songkhla
Glossiphoniid leeches are characterized as the only annelids that have parental care behavior by carrying cocoons and juveniles directly on the ventral surface for protection and feeding (
Placobdelloides siamensis (Oka, 1917) is the only leech species currently reported from several different turtles of the family Geoemydidae in Thailand, which commonly inhabit flowing-water ecosystems (
This study presents the first report of the use of a combination of morphological and molecular techniques to describe a new leech species that parasitizes Asian leaf turtles, Cyclemys dentata (Gray, 1831) and dark-bellied leaf turtles, C. enigmatica Fritz et al., 2008. This newly discovered turtle leech is here presented along with new information about its identification and geographic distribution in Thailand.
Leech specimens were collected from two different turtle species at six different collecting sites. Seven leaf turtles (three individuals of C. dentata and four individuals of C. enigmatica) were collected from the bottom of small muddy puddles or patches of approximately 20–30 cm depth in rubber plantations in Sadao District, Songkhla Province (6°62'57.7"N, 100°41'12.7"E) on 21 October 2018. Leeches were removed from the body and shell of each turtle using forceps and then stored in sealed bottles with water from the capture sites to keep them alive. The carapace length was measured for all turtles, after which they were released back into their capture sites when finished.
Leeches were maintained in a glass container (10×12×8 cm3) half full of puddle water and fitted with an oxygen-pumping machine for behavioral study in the laboratory. Afterward, some individuals were preserved in absolute ethanol in a relaxed stage for scanning electron microscopy (SEM) and molecular techniques, while still others were preserved in 70% ethanol in a relaxed stage for identification.
Each specimen was examined for eye number and placement, annulation, digestive system (including the number and structure of gastric ceca), and reproductive system, following
The leech specimens in absolute ethanol were sectioned into two equal pieces. The posterior part was used for DNA extraction with TIANamp Genomic DNA Kit (catalog number DP304-02; TIANGEN Biotech (Beijing) Co., Ltd., Beijing) while the anterior part was stored in absolute ethanol to be used later for a DNA sample stock. For the proteinase K treatment step, tissue samples were lysed for two hours at 58°C. The DNA was eluted from the spin column with 200 µl of buffer.
Polymerase chain reactions (PCR) were prepared using the EP0402 TAQ DNA POLYMERASE. Two mitochondrial gene fragments were amplified namely, cytochrome c oxidase subunit I (CO-I) and nicotinamide adenine dinucleotide dehydrogenase subunit I (ND-1) following
GenBank accession numbers for leech sequences used in the phylogenetic analysis of Placobdelloides.
Taxon | Locality | GenBank accession numbers | |
---|---|---|---|
COI | ND1 | ||
Ingroup | |||
Placobdelloides sirikanchanae sp. nov. | Songkhla, Thailand | MK282428 | MK282433 |
Songkhla, Thailand | MK282429 | MK282434 | |
Songkhla, Thailand | MK282430 | MK282435 | |
Songkhla, Thailand | MK282431 | MK282436 | |
Songkhla, Thailand | MK282432 | MK282437 | |
Placobdelloides jaegerskioeldi (Johansson, 1909) | Sudan, South Africa | AY962463 | AY962450 |
Placobdelloides multistriatus (Johansson, 1909) | Louisiana, USA | DQ414338 | DQ414383 |
Placobdelloides siamensis (Oka, 1917) | Bangkok, Thailand | AY962449 | AY962462 |
Bangkok, Thailand | MH777415 | MH777409 | |
Bangkok, Thailand | MH777416 | MH777410 | |
Bangkok, Thailand | MH777417 | MH777411 | |
Bangkok, Thailand | MH777418 | MH777412 | |
Bangkok, Thailand | MH777419 | MH777413 | |
Bangkok, Thailand | MH777420 | MH777414 | |
Udon Thani, Thailand | MN221458 | MN242784 | |
Udon Thani, Thailand | MN221459 | MN242785 | |
Udon Thani, Thailand | MN221460 | MN242786 | |
Outgroup | |||
Alboglossiphonia heteroclita (Linnaeus, 1761) | Michigan, USA | AF116016 | AY047339 |
Alboglossiphonia quadrata (Moore, 1949) Sawyer, 1986 | Namibia, South Africa | AY962455 | AY962441 |
Alboglossiphonia weberi (Blanchard, 1897b) | Hawaii, USA | AY962453 | AY962440 |
Batracobdelloides tricarinata (Blanchard, 1897a) | Hoedspruit, South Africa | AY962457 | AY962445 |
Glossiphonia baicalensis (Stchegolew, 1922) | Lake Baikal, Russia | AY047329 | AY047355 |
Glossiphonia complanata (Linnaeus, 1758) | United Kingdom | MF458715 | AY047345 |
Glossiphonia concolor (Apathy, 1888) | Kila River, Sweden | AY962458 | AY962446 |
Glossiphonia elegans (Verrill, 1872) | Connecticut, USA | AY047322 | AY047335 |
Glossiphonia verrucata (Müller, 1844) | Rio s’ Adde, Italy | AY962459 | AY962447 |
Helobdella fusca (Castle, 1900) | Michigan, USA | AF329038 | AF329061 |
Helobdella robusta (Shankland, Bissen & Weisblat, 1992) | Sacramento River, USA | MF067148 | MF067201 |
Hemiclepsis marginata (Müller, 1774) | Étang de la Musse, France | AF003259 | AY047336 |
Hirudo medicinalis (Linnaeus, 1758) | Gotland, Sweden | HQ333517 | KU672396 |
Marsupiobdella africana Goddard & Malan, 1912 | South Africa | AF116015 | AY047347 |
Placobdella montifera (Moore, 1906) | Washington, USA | MF067129 | MF067212 |
Placobdella pediculata (Hemingway, 1908) | Lake Pepin, USA | MF067121 | MF067222 |
Theromyzon bifarium Oosthuizen & Davies, 1993 | North USA | AY047330 | AY047356 |
Theromyzon tessulatum (Müller, 1774) | Europe | AY047318 | AY047338 |
The DNA sequences were aligned using ClustalW v. 1.83 (
The maximum likelihood analysis consisted of 2000 tree search replicates, with 25 initial GAMMA rate categories and final optimization using four GAMMA shape categories. Bootstrap values were calculated using 2000 pseudoreplicates of the rapid bootstrap algorithm. Bayesian analysis was run for 20 million generations with trees sampled every 100 generations with a general time reversible (GTR) model and GAMMA distribution of nucleotide rates for all partitions. Burn-in was set to 10%. Bootstrap values ≥70% for maximum likelihood analysis and Bayesian posterior probabilities of ≥95% were considered a priori as being indicators of highly supported nodes (
In total, six muddy puddles on rubber plantations (6°62'57.7"N, 100°41'12.7"E) were inhabited by two turtle species: Cyclemys dentata and C. enigmatica (Figure
Placobdelloides multistriata (Johansson, 1909) by original designation.
eyes one pair, esophageal organ, crop caeca seven pairs, mouth pore terminal (
Placobdelloides species can be found in Africa (P. fimbriata (Johansson, 1909); P. jaegerskioeldi (Johansson, 1909); P. multistriata (Johansson, 1909)), Australia and United States, eastward to India (P. fulva (Harding, 1924); P. emydae (Harding, 1920); P. undulata (Harding, 1924); P. horai (Baugh, 1960); P. indica (Baugh, 1960)), Southeast Asia (P. siamensis in China and Thailand; P. okadai (Oka, 1925) in China; P. okai (Soós, 1969); P. stellapapillosa
Holotype (ZMKU-ANN-0006), puddle on rubber plantation, Sadao District, Songkhla Province, Thailand (6°62'57.7"N, 100°41'12.7"E), 21 October 2018. Paratypes (nine individuals, ZMKU-ANN-0007 to 0015), same locality data as the holotype. All collected specimens were kept in 70% alcohol and deposited at the Zoological Museum of Kasetsart University (ZMKU), Department of Zoology, Faculty of Science, Kasetsart University (13°50'53.6"N, 100°33'47.3"E) on 23 November 2018.
This species can be recognized from its elongated, narrow body, crimson median dorsal line, rich dark green pigmentation, 13–17 well-developed knob papillae on each annulus, symmetrical dorsal papillae between the left and right body sides, male gonopore on XIa1/a2, female gonopore on XIa3/XIIa1, amorphous salivary glands, smooth surface with random pits inside the anterior sucker, and rugged surface with randomly distributed pits inside the posterior sucker.
External morphology. A mature Placobdelloides sirikanchanae sp. nov. (ZMKU-ANN-0006) has an elongated, dorso-ventrally flattened, tri-annulate body (Figure
Annulation. Somites I-III are uni-annulate, IV and V are biannulate (annuli 4–7), VI-XIV are tri-annulate (annuli 8–34), XV-XVIII are uni-annulate (annuli 35–38), XIX-XXV are tri-annulate (annuli 39–59), XXVI is biannulate (annuli 60–61), and XXVII-XXXIV are uni-annulate (annuli 62–69).
Digestive system
: A cylindrical slender proboscis resides in a membranous sheath that protrudes through the lip of the posterior subterminal mouth (Figure
Reproductive system. The male gonopore rim is thick and curled. The ejaculatory bulb on XIa2-XIIa2 (annuli 24–27) is an apple-like sac opening into the vas deferens. Two vas deferens extend posteriorly and recurve in front of post ceca anteriorly to connect to the testisacs. Six pairs of ovoid testisacs are present, and each is located in the space between a pair of crop cecae. The female gonopore rim is thinner and smoother than that of the male. The spermatheca is a rectangular sac on XIIa2-XIIIa3 (annuli 27–31), which opens into bifurcated ovisacs.
External morphology. The average relaxed body length is 10.77 mm long (range 7.62–40.39 mm, N = 20), and the average relaxed body width at the widest point (annuli 35, XV) is 3.96 mm (range 3.52–4.89 mm, N = 20). The average anterior sucker diameter is 1.08 mm (range 0.93–1.42 mm, N = 20). The average posterior sucker diameter is 1.94 mm (range 1.70–2.60 mm, N = 20), half the size of the maximum body width.
Color
in life is uniformly dark brown to greenish, with randomly distributed dark brown, red, yellow, and especially rich dark green pigments. There is a crimson median line present dorsally from the neck region to the posterior sucker (Figure
Posterior part of live specimens A holotype Placobdelloides sirikanchanae sp. nov. showing dorsal crimson median line (yellow arrow) with numerous scattered dark green pigments. Bubbles are emerging from the respiratory pores (white arrows) B Placobdelloides siamensis (Oka, 1917) from Bangkok, Thailand showing yellow median line (yellow arrow) with numerous scattered yellow pigments.
Reproductive system. The length of the ovisacs depends on the reproductive stage. During the normal, non-reproductive period, ovisacs are present on XIIIa1-XIVa1 (annuli 29–32), but they can extend from XIIIa1 to XXa1 (annuli 29 to 42 (4th pair of crop cecae)) during the gestational period.
Molecular comparisons based on p-distances among five specimens of P. sirikanchanae sp. nov. from a rubber plantation in the Sadao District, Songkhla Province, Thailand revealed a difference of 2.5–6.2% for 518 nucleotides of COI (GenBank MK282428–MK282432) and 1.3–3.3% for 555 nucleotides of ND1 (GenBank MK282433–MK282437) (see Tables
P-distance values of COI genes within (diagonal) and among 4 species of Placobdelloides including P. sirikanchanae sp. nov. identified in this study.
Species | 1 | 2 | 3 | 4 |
---|---|---|---|---|
1 Placobdelloides sirikanchanae sp. nov. | 2.5–6.2% | |||
2 Placobdelloides siamensis | 10.4–27.7% | 0.0–10.1% | ||
3 Placobdelloides multistriatus | 19.3–21.7% | 15.6–30.6% | – | |
4 Placobdelloides jaegerskioeldi | 21.0–23.5% | 17.3–31.6% | 12.6% | – |
P-distance values of ND1 genes within (diagonal) and among 4 species of Placobdelloides including P. sirikanchanae sp. nov. identified in this study.
Species | 1 | 2 | 3 | 4 |
---|---|---|---|---|
1 Placobdelloides sirikanchanae sp. nov. | 1.3–3.3% | |||
2 Placobdelloides siamensis | 5.4–6.9% | 0.0–1.7% | ||
3 Placobdelloides multistriatus | 15.1–15.8% | 15.1–15.4% | – | |
4 Placobdelloides jaegerskioeldi | 15.1–16.0% | 13.4–13.6% | 14.3% | – |
Dark-bellied leaf turtles (Cyclemys enigmatica).
Asian leaf turtles (C. dentata).
Placobdelloides sirikanchanae sp. nov. can be found attached on the shell surface, both the carapace and plastron, of C. dentata and C. enigmatica, which inhabit the bottom of enclosed shallow muddy puddles on rubber plantations. In the rainy season, several puddles will be connected due to an increase in the water level. Numerous small vertebrates are present in these puddles, such as small fishes or tadpoles. In the dry season, the puddles will be disconnected as the shallower waters disappear from evaporation. These aquatic ecosystems usually have low oxygen due to decomposition of leaf litter and nonflowing water.
Ten individuals of P. sirikanchanae sp. nov. were released into a tank with water from the type locality and equipped with an oxygen pump. All ten died almost immediately. The ten remaining specimens survived in a sealed bottle under low dissolved oxygen conditions. No ventilation (undulating movement display) was observed. After three days, they initiated copulation and deposited eggs in the sealed bottles.
Ten individuals of P. sirikanchanae sp. nov. displayed reproductive activity in a sealed bottle (low oxygen condition). One copulated with another individual for a few hours before they separated. The beginning of gestation was observed inside the ovisacs of both individuals (seen through the ventral surface) 2–3 days after copulation and gestation continued for approximately 3–4 days more before deposition of eggs. Round creamy-colored eggs, approximately 104–115 eggs per individual, were deposited and aggregated inside the transparent membrane beneath the venter groove of the parent (Figure
The species is named in honor of Associate Professor Prapaisiri Sirikanchana, the pioneer aquatic parasitologist of Thailand. The following common names, Sirikanchana’s leech (English), Pling Arjan Prapaisiri (Thai), and Sirikanchanas Plattegel (German) are suggested.
Placobdelloides sirikanchanae sp. nov. was distinguished from P. siamensis (based on the original description by
Comparison of morphological characters, egg number per clutch, host, and distribution of Placobdelloides sirikanchanae sp. nov. and P. siamensis (Oka, 1917) in Thailand.
Characters | P. sirikanchanae sp. nov. | P. siamensis (Oka, 1917) | |
---|---|---|---|
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|
||
Host | Cyclemys dentata and C. enigmatica | Siebenrockiella crassicollis | Cuora amboinensis, Heosemys annandalii, Malayemys macrocephala, M. subtrijuga, M. khoratensis, and S. crassicollis |
Distribution | Sadao, Songkhla | Lampam, Pattalung | Bangkok and Udon Thani |
Maximum relaxed length (mm) | 40.39 | 15.00 | 25.00 |
Maximum relaxed widest width (mm) | 4.89 | 4.00 | 5.57 |
Body shape | Elongated narrow | Elongated oval | Elongated oval |
Eye location | III | III | III |
Anterior sucker diameter (mm) | 1.08 | 2.50 | 1.86 |
Anterior sucker surface | Smooth with numerous pits | – | Smooth with numerous pit |
Position of proboscis opening | Posterior subterminal | Posterior subterminal | Posterior subterminal |
Number of dorsal papillae on each annulus | 13–17 | 22–27 | 5–9 |
Shape of dorsal papillae | Well-developed knob shape | Cone shape | Well-developed longitudinal rod shape |
Total annuli | 69 | 67 | 69 |
Live dorsal color | Dark brown greenish with crimson median line | Uniform gray with faint brown median line (in alcohol) | Brownish gray with yellow median line and four pairs of scarlet dots |
Male gonopore location | XIa1/a2 (annuli 23/24) |
XIa3/XIIa1 (annuli 25/26) |
Xa3/XIa1 (annuli 22/23) |
Ejaculatory bulb | Apple-like sac | – | Glasses-like sac |
Female gonopore location | XIa3/XIIa1 (annuli 25/26) |
XIIa2/a3 (annuli 27/28) |
XIa2/a3 (annuli 24/25) |
Spermatheca | Rectangular sac | – | Slender sac |
Anus location | Between last annuli and posterior sucker | Between last annuli and posterior sucker | Between last annuli and posterior sucker |
Posterior sucker diameter (mm) | 1.94 | 3.00 | 3.00 |
Posterior sucker surface | Rough with random scattered pits | – | Smooth with random scattered pits |
Eggs per clutch | 104–115 | – | 173–412 |
Placobdelloides sirikanchanae sp. nov. was identified as a new leech species based on morphological and genetic characteristics and was shown to be distinct from other members of its genus. Comparison of P. sirikanchanae with other species of Placobdelloides that parasitize crocodiles and turtles revealed the following: P. bancrofti is distinguished from P. sirikanchanae by having one annulus separating the male and female gonopores and an absence of dorsal papillae; P. emydae has a slightly dilated head and three pairs of metameric papillae on the dorsum; P. fimbriata has a unique gill-like marginal fringe; P. multistriata has two pairs of salivary glands and the absence of dorsal papillae; the original description of P. siamensis from the description by
The phylogenies (Fig.
This is the first report of the reproductive biology of P. sirikanchanae. This hermaphroditic leech displayed monandrous copulation and exchanged pseudospermatophores with other leeches a few hours before separation . The gestational period after copulation through egg deposition was approximately 5–7 days, which began in the ovisacs beginning 2–3 days after copulation. The family Glossiphoniidae is unique in that members of this family exhibit parental care of their eggs and juveniles (
This is the first report of P. sirikanchanae parasitizing Asian leaf turtles (C. dentata) and dark-bellied leaf turtles (C. enigmatica). In the field surveys of this study, both the leech and the turtles inhabited the bottom of enclosed shallow muddy puddles or patches in rubber plantations. Small puddles and patches are a temporary aquatic system that usually occurs after rain and disappears within a few weeks or months from evaporation or seeping into the ground. In addition, this aquatic system usually has low dissolved oxygen conditions from leaf decomposition and the absence of flowing water, but despite this, there were numerous small vertebrates living there, such as fishes and Rhacophorus tadpoles (
For P. sirikanchanae, its small clutch size and faster development times might be an adaptation to living in these temporary ponds. Moreover, the observed behavior in the laboratory combined with water conditions in the field indicated that P. sirikanchanae is a leech that can tolerate low dissolved oxygen conditions.
Cyclemys dentata and C. enigmatica are members of the family Geoemydidae, the main freshwater turtle family found in Thailand, along with Cuora amboinensis, Heosemys annandalii, Malayemys macrocephala, M. subtrijuga, M. khoratensis, and Siebenrockiella crassicollis, all of which are the hosts of P. siamensis (
This work was supported by the Human Resource Development in Science Project (Science Achievement Scholarship of Thailand, SAST), Center of Excellence on Biodiversity (BDC), Office of Higher Education Commission (Project Code BDC-PG2-161004) and the Department of Zoology, Faculty of Science, Kasetsart University. The authors would like to thank Mrs. Pooncharat Trivalairat for field guidance and collecting some of the examined leech specimens. This research was approved by the Institutional Animal Care and Use Committee, Faculty of Science, Kasetsart University, Thailand under project number ACKU61-SCI-028.